The cytolethal distending toxin (CDT) is a potent bacterial toxin produced by a growing list of unrelated bacterial pathogens including scattered isolates of E. coli and Shigella isolates, Campylobacter spp., Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enteropathogenic Helicobacter spp. Initially, CDT was characterized by its capacity to induce massive cellular distension and cell death. Cells treated with CDT undergo an irreversible block in cell division caused by disruption of the cell cycle in G2. The specific events leading to CDT-mediated growth arrest parallel those following induction of the mitotic DNA damage checkpoint. CDT is the product of three genes designated cdtA, cdtB, and cdtC, which encode proteins with molecular masses of 30, 32, and 20 kDa. Genetic and biochemical evidence indicate that all three polypeptides are required for cellular intoxication. We recently reported that CdtB bears sidnificance position-specific sequence relatedness to mammalian type I DNase. Mutational analysis indicates that the DNase-related active site residues in CdtB are required for biological activity. In preliminary studies show that purified CdtB possesses Mg2plus- dependent DNase activity. We also present evidence indicating that CDT damages chromosomal DNA followed by activation of elements of the DNA damage checkpoint cascade. Although not toxic when added alone to cells, introduction of CdtB into cells results in the entire spectrum of CDT activities. We therefore hypothesize that CdtB mediates the cytolethal effects of CDT while CdtA and CdtC are required for cell binding and/or translocation of CdtB. In this application we propose to: 1) define the role of each of the three CDT polypeptides in cell binding and CdtB entry, 2) determine the mechanism by which CdtB traffics through the cell and translocates into nucleus (the apparent site of CdtB action, and 3) define the series of events leading to CDT-mediated growth arrest and death. Completion of these aims will provide new insights into the novel action of this potent bacterial toxin.